Antisense therapy involves the use of oligonucleotides having complementary sequences to target RNA or DNA. The antisense oligonucleotide binds to the target RNA or DNA. Upon binding to the target RNA or DNA, the antisense oligonucleotide can selectively inhibit the genetic expression of these nucleic acids or can induce some other events such as destruction of a targeted RNA or DNA or activation of gene expression. Destruction of targeted RNA can be effected by RNase H activation or by linking strand cleavers to the oligonucleotide.
One class of oligonucleotides that have been synthesized are the 2'-O-substituted oligonucleotides. Such oligonucleotides have certain useful properties. In U.S. patent application Ser. No. 814,961, filed Dec. 24, 1991, now abandoned, entitled Gapped 2' Modified Phosphorothioate Oligonucleotides, assigned to the same assignee as this application, the entire contents of which are herein incorporated by reference, 2' substituted nucleotides are introduced within an oligonucleotide to induce increased binding of the oligonucleotide to a complementary target strand while allowing expression of RNase H activity to destroy the targeted strand. In a recent article, Sproat, B. S., Beijer, B. and Iribarren, A., Nucleic Acids Research, 18:41 (1990), the authors noted further use of 2'-O-methyl substituted oligonucleotides as "valuable antisense probes for studying pre-mRNA splicing and the structure of spliceosomes".
The advent of automated DNA synthesizers has resulted in ease of synthesis of oligonucleotides having specific sequences of choice. Oligonucleotides are synthesized utilizing nucleotide precursors. These nucleotides, in turn, are synthesized utilizing nucleoside precursors. 2'-O-methyl nucleosides are known; indeed the 2'-O-methyl ether of the four major ribonucleotides occur as minor components in natural RNA. Robins, M. J., Naik, S. R. and Lee, A. S. K., J. Org. Chem., 39:1891 (1974) reported a low yield synthesis of 2'-O- and 3'-O-methyl guanosine via a stannous chloride catalyzed monomethylation by diazomethane. As was later reported by Robins, M. J., Hansske, F. and Bernier, S. E., Can. J. Chem., 59:3360 (1981), "convenient and high yield methods have been devised for synthesis of the 2'-O- and 3'-O-methyl ethers of adenosine, cytidine, and uridine . . . However, guanosine has presented significant difficulties." In the foregoing paper, the authors reported an improved synthesis of 2'-O and 3'-O-methyl guanosine. The synthesis was improved by effecting the stannous chloride catalyzed diazomethane methylation of 2,6-diamino-9-(.beta.-D-ribofuranosyl)purine (2-aminoadenosine) in place of guanosine. The diamino purine moiety was then reduced to the corresponding guanine moiety with adenosine deaminase. In a further diazoation reaction described by Singer and Kusmierek, Biochemistry 15: 5052 (1976), a mixture of 2' and 3'-O-ethyl guanosine was reported to result from the treatment of guanosine with diazoethane. The alkylation also resulted in alkylation of the heterocyclic base. The alkylated product was treated with base to remove the ethyl group from the heterocyclic base. The resulting product was identified by virtue of having the same UV spectrum as that of guanosine, but a Rf differing from the Rf of guanosine.
A further improvement in the synthesis of 2'-O-methyl nucleosides was reported by Inoue, H., Hayase, Y. Imura, A., Iwai, S., Miura, K. and Ohtsuka, E., Nucleic Acids Research, 15:6131 (1987). This method of synthesis was effected utilizing CH.sub.3 I in the presence of Ag.sub.2 O. This method proved useful for all of the common nucleotides with the exception of guanosine. As reported by these authors, guanosine proved refractory to this synthetic method. Thus these authors again had to effect the 2'-O-methylation of guanosine with diazomethane. In order to avoid methylation of the amino functionality of the guanine base moiety, the guanine base moiety was blocked with an isobutyryl group. Additionally, to avoid methyl esterification of the 3'-O functionality of the sugar moiety, a TIPDS (tetraisopropyldisiloxane) blocking group was used to block both the 3' and the 5' hydroxyls of the sugar moiety.
The most recent investigators to address the synthesis of 2'-O-methyl guanosine (and 2'-O-allyl guanosine) are Sproat et al., supra and Sproat, B. S., Iribarren, A. M., Garcia, R. G. and Beijer, B., Nucleic Acids Research, 19:733 (1991). In both of these Sproat et al. publications, the investigators presented a further synthetic pathway to 2'-O-methylguanosine and 2'-O-allylguanosine. They characterized the further pathway with respect to the prior known synthetic methods as "avoids(ing) . . . the use of the highly toxic and potentially explosive reagent diazomethane" and being "far superior to the use of silver oxide/methyl iodide." This same synthetic method of the Sproat et al. investigators is also published in B. S. Sproat and A. I. Lamond, "2'-O-Methyloligoribonucleotides: synthesis and applications," Oligonucleotides and Analogues, ed. F. Eckstein, (IRL Press, 1991). While avoiding the use of diazomethane, when applied to guanosine this latest Sproat et al. synthetic pathway requires six steps to achieve a guanosine compound. Of the six steps in this synthetic pathway, five require separate chromatographic purifications.
A further 2'-O-alkylated guanosine compound is known. This compound, 2'-O-methylthiomethylguanosine, was reported by Hansske, F., Madej, D. and Robins, M. J., Tetrahedron, 40:125 (1984). It was produced as a minor by-product of an oxidization step during the conversion of guanosine to 9-.beta.-D-arabinofuranosylguanine, i.e. the arabino analogue of guanosine. The addition of the 2'-O-methylthiomethyl moiety is an artifact from the DMSO solvent utilized during the oxidization procedure. The 2'-O-methylthiomethyl derivative of 2,6-diaminopurine riboside was also reported in the Hansske et al. publication. It was also obtained as an artifact from the DMSO solvent.
It is an object of this invention to provide methods of synthesis of 2'-O-alkylated guanosine and guanosine analogues that avoids the use of diazomethane.
It is a further object of this invention to provide methods of synthesis of 2'-O- and 3'-O-alkylated 2,6-diaminopurine riboside compounds.
It is an additional object of this invention to provide efficient syntheses for 2'-O-alkylated guanosine and guanosine analogs.